Find Your Free Essay Examples

In This Colorimetric Aptasensor

In this colorimetric aptasensor, two assay strategies including adsorbed thiolated aptamer(T-Apt)and adsorbed non-thiolated poly-Adenine aptamer(polyA Apt) on the gold nanoparticles (AuNPs) surfac were used for a simple, rapid and accurate determination of ampicilline(AMP). In this methods by incubating the AuNPs and the aptamer prior to target addition T-Apt and poly-A Apt by difference mechanism adsorbed on the gold nanoparticles and a nano-bio conjugation was formed. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. The sensing methods was developed to produce a remarkable optical absorbanc difference in the absence and presence of AMP. The proposed assay selectively recognized AMP in the presence of other interfering substances with the same chemical structure as AMP and thus, can be applied to real samples for the rapid screening of AMP. In adsorbed T-Apt ampicillin colorimetric assay, detection of ampicillin was linear in the concentration range 1-600 nM with 0.1 nM as the limit of detection and in adsorbed polyA Apt ampicillin colorimetric assay, detection of ampicillin was linear in the concentration range 1-400 nM with 0.49 nM as the limit of detection.

Keywords: Aptasensor; non-thiolated aptamer, ampicillin; polyA aptamer, gold nanoparticles

1. Introduction

An antibiotic is a type of antimicrobial substance active against bacteria and used for treatment of human and poultry diseases. However misuse of antibiotics in food industry may caused undesired levels of residues in food supplies[1-3] and endanger the food safety, thus cause human health hazards [4, 5]. Moreover, aberrant prescribing of antibiotics leading to antibiotic resistance. If antibiotic resistance to grow, the antibiotics used to treat infections today will become ineffective or will stop working altogether in the future. Therefore accurate quantitative detection of antibiotics (such as AMP) is very important in generation of healthy food-producing animals and human health. AMP is one of the most commonly used β-lactam antibiotics. It treat a number of bacterial infections, such as respiratory tract infections, ear infections, urinary tract infections, meningitis, salmonellosis, and used for eradicated of bacterial infections (urogenital and skin) in food-producing animals [6, 7].

Over the past few years, various methods for detection of AMP have been developed. This methods including High-performance liquid chromatography[8], Liquid Chromatography Tandem Mass Spectrometry[9], colorimetry[10, 11], Fluorescent Immunoassay [12], electro chemiluminescence[13] and electrochemistry[14, 15]. These common methods suffer from some defects, they are time consuming and complicated, require skilled user, expensive and elaborate instrumentation which limited them as the on-site assays in practical applications. So it is highly required that a simple, fast and practicable method for the detection of AMP residues in food products and human body with favorable selectivity and sensitivity. Aptamers are single-stranded DNA or RNA molecules capable of tightly binding to specific targets. Aptamers have key advantages over antibodies including more chemical stability, simpler synthesis/modification, easier storage, better reproducibility, lower cost, and especially wider range of recognizable targets from metal ions, small molecules to macromolecules (such as proteins), and even viruses and cells[16, 17]. These privileges have made them a powerful and versatile functional molecular tool for a various applications including medical applications [18-21], bio-imaging[20] food inspection[21, 22] hazard detection[23, 24], biosensors[25 -29]. In the field of biosensor development, aptamers are especially advantageous for detection of small molecules because they enable unique techniques for signal generations that are not applicable with antibodies [30]. Due to antibiotics like other small molecules cannot act as an antigen independently, high specificity and affinity antibodies are difficult to prepare, therefore use of aptamer is preferable to antibody in molecular recognition systems. In aptamer-based biosensors (aptasensors), aptamer was active in different sensing analysis such as optical and electrochemical. Colorimetric method has been used extensively due to its low cost, simplicity, practicality and particularly observation of the color change by the naked eyes [31]. Nanomatrials have revolutionized in molecular sensing tools. Particularly, the AuNPs possess unique optoelectronic properties, which can readily be tuned by varying their size, shape, and the surrounding chemical environment [32]. AuNPs possess a strong SPR band that results from the spatial length reduction of electronic motion and the coherent oscillation of the electrons. In addition, the SPR band of AuNPs has strong distance-dependent properties, whereby the aggregation of AuNPs of appropriate sizes (diameter >3.5 nm) can induce interparticle surface plasmon coupling, resulting in a visible color change from red to blue [33]. Thus, AuNPs provide a practical platform for colorimetric sensing of any target analyte.

Typically in design and construction of colorimetric aptasensor two strategies including free[34, 35] and adsorbed[36-38] aptamer were used. In the free aptamer strategy, the aptamer and target were incubated to allow binding followed by exposure to the gold nanoparticles (AuNPs). Interactions between the non-bound analytes and the AuNPs surface resulted in a number of false positives and therefore lead to reduction of sensitivity of this colorimetric method. Currently, one of the best methods to conjugate aptamars on gold nanoparticles surface using thiol modified aptamers[39]. However studying non-thiolated DNA is important for several reasons[40]. First, The cost of synthesis of a typical aptamer is 90% less than thiolated counterpart. Second, fundamental understanding of the interaction between DNA and gold can be further increased. Finally, new applications may be derived from this hybrid material including biosensor development[41-43] control of enzymatic reaction[44], nanoparticle synthesis[45], and medicine[46]. Ideally, non-thiolated DNA should adopt an upright conformation just like the thiolated counterpart to achieve molecular recognition[40].

The main purpose of this study, comparison of the the role of two methodology adsorbed Apt including T-Apt and PolyA Apt, on AuNPs surface on performance of colorimetric aptasensor to detect AMP. Here in we used two methods to adsorb Apt on AuNPs surface, in the first method T-Apt conjugated to AuNPs, while in the second method polyA modification-free Apt conjugated to AuNPs prior to target addition. The adsorbed aptamer was able to bind the target while preventing non-specific interactions.

2. Experimental section

2.1. Chemicals and materials

The synthetic anti-ampicillin oligonucleotides, single stranded DNA (ssDNA) (5′- (SH)-(CH2)6-GCGGGCGGTTGTATAGCGG-3′), 5′-GCGGGCGGTTGTATAGCGG(T)15-(A)12-3′ were obtained from Bioneer Co., Ltd.( Daejeon, south korea). Hydrogen tetrachloroaurate(III), trisodium citrate, polydiallyldimethylammonium chloride(PDDA), dithiothreitol (DTT) , ethyl acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), citrate and ampicillin, amoxicillin, penicillin, benzylpenicillin and lincomycin obtained from Sigma Aldrich. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance>18.2 MΩ. The glassware was rinsed with aqua regia and rinsed with ultrapure water and dried before use.

2.2. Instrumentation

The UV-Visible measurements were recorded on BioTek Synergy HTX Multi-Mode Reader. TEM analyses were carried out using Zeiss EM10C microscope.

2.3. Synthesis of AuNPs

A 100 mL solution of 1 mM HAuCl4 was heated at its boiling point with stirring, and 10 mL of a 38.8 mM sodium citrate solution was added. The solution continued to boil with mixing for 20 min. The sample was cooled to room temperature and were stored in dark bottle at 4°C for further use. The final AuNPs concentration was determined to be 10 nM based on the extinction measured at 520 nm, using ε=2.4×108 L. mol-1.cm-1.

2.4. Preparation of T-aptamer-modified AuNPs (T-Apt-AuNPs) probe

100 µM aptamer heated at 90°C for 3 min and then cooled at 4°C for 1 hour. This heating and cooling step helps to maintain the structural flexibility of the aptamers (for binding AMP). The 5′-thiol-modified oligo-nucleotides were received in a disulfide form [HOCH3(CH2)5S-S-5′-oligo], protected by a mercaptohexanol group. Herein, DTT is a reducing agent, which is intended to disrupt any disulfide bonds (-S-S-) and ensures that the free SH groups are ready to react with the gold surface [39]. 10 µL DTT solution (1.0 N) and 50 µL(50 µM) aptamer were mixed together, vortex, then kept at room temperature half an hour to ensure the disulfide cleavage finished completely. Remove excess DTT and unwanted thiol fragments from the thiol-modified oligonucleotide mixture by extracting with ethyl acetate 3 times, using 50 µL per extraction. Discard the upper layer after vortexing the mixture. The lower layer is T-Apt that ready to react directly to gold nanoparticles. Anti-AMP Apt was reacted directly with the AuNPs through attachment of the oligo-thiol units onto the AuNPs surface. Afterwards, 40 µL AuNPs and 10 µL 0.5 µM T-Apt were mixed together and kept at room temperature for overnight. During this time, the aptamers were wrapped in AuNPs by strong covalent bond between SH and gold.

2.5. Preparation of polyA aptamer-modified AuNPs (polyA Apt-AuNPs)

First, a small volume (i.e., 1-3 µL) of poly A- tailed DNA stock solution (100 µM in 5 mM HEPES buffer, pH 7.6) was added into 250 µL of as-synthesized AuNPs solution (10 nM) and mixed by a brief vortex. Second, pH 3 citrate buffer was added into the AuNPs solution to make a final citrate concentration of 10 mM. After 3 min incubation at room temperature, the pH of the AuNPs solution was adjusted back to neutral by adding 500 mM HEPES buffer to a final HEPES concentration of 30 mM (pH 7.6). The sample was incubated for another 5-10 min. Finally, the DNA-AuNPs mixture was centrifuged at 15,000 rpm and the supernatant was removed. The pellet was washed with 5 mM HEPES to remove free DNA. This centrifugation and washing procedure was repeated 4-5 times to ensure complete removal of free DNA and the conjugate was dispersed in 5 mM HEPES buffer for further use [40].

2.6. Adsorbed T-Apt ampicillin colorimetric assay

10 µL AMP added to the T-Apt modified AuNPs solution (50 µL) to make the final concentration of AMP ranging from 0¬–¬¬¬600 nM. After 1 h, PDDA to final concentration of 20 nM was added to the assay solution, and after 5 min the absorption spectra (the wavelength range from 400 to 800 nm) and absorbance values at 520 nm (A520) and 680 nm (A680) was recorded.

2.7. Adsorbed polyA Apt colorimetric assay

10 µL AMP added to the polyA Apt-AuNPs solution (50 µL) to make the final concentration of AMP ranging from 0¬–¬¬¬600 nM. After 1 h, PDDA to final concentration of 20 nM was added to the assay solution, and after 5 min the absorption spectra (the wavelength range from 400 to 800 nm) and absorbance values at 520 nm (A520) and 680 nm (A680) was recorded.

3. Results and discussion

3.1. AMP sensor design

The fundamental molecular interaction between aptamers and their target is largely due to hydrogen bonding, electrostatic interactions, stacking of aromatic rings and van der Waals interaction[47]. In the presence of the target, aptamer is transformed into its 3-dimensional structure to interact fully with their target. The chemical structure of ampicillin with the secondary structure of ssDNA ampicillin aptamer shown in Fig 1(a). Ampicillin is a β-lactam antibiotic, and is characterized by a β-lactam ring in its molecular structure. Its side chain (circled) contains a primary amine functional group and a benzene ring. Interestingly, the ampicillin aptamer was reported to be highly specific to the ampicillin side chain[48]. The aptamer sequence responsible for the binding of ampicillin was also reported to be “-GGT(T)-” in the loop region as well as “-GC-” base pairing (circled) at the joint of a loop and stem[48].

The strategy for the colorimetric detection of AMP for both mentioned design( T-Apt and polyA Apt) is illustrated in Fig 1. As shown in Fig 1 (b), in the absence of AMP, T-apt is absorbed on the surface of AuNPs by strong covalent bond between SH and gold [39]. Similarly non-thiolated DNA can also be attached via through amine groups of DNA base adsorption [49, 50]. PolyA Apt is absorbed on the surface of AuNPs by specific noncovalent nucleotide adsorption. A block of poly-adenine (poly-A) could be used to achieve a high density of DNA attachment. Since adenine is adsorbed with a higher affinity than thymine, a straightforward method is to make one end poly-A. The thymine chain is used as a spacer to keep aptamer configuration which is very essential for the strong interaction between the aptamer and it,s target. poly-A block to serve as an anchor on AuNPs (e.g. a replacement of the thiol functionality) Fig 1 (c). So AuNPs are stabilized by the aptamer(T-Apt or polyA Apt) against PDDA-induced aggregation. However, when AMP exists, it combines with Apt specifically, which removes the ssDNA layer from AuNPs competitively. Then the color of the solution turns from red to purple due to the AuNPs aggregation, according to AMP concentration.

3.2. Optimization of the reaction conditions

3.2.1. Optimization of the reaction conditions for adsorbed T-Apt colorimetric assay

To optimize PDDA concentration, various PDDA concentrations i.e. 0, 3, 5, 10, 15, 20, 30 and 40 nM were incubated with 10 nM AuNPs for 15 min. It was observed that 20 nM PDDA was sufficient enough to aggregate the AuNPs and change the red color of the AuNPs to blue (Fig. 1S). Hence, 20 nM PDDA was used for all the experiments. After optimaization of PDDA concentration, aptamer concentration was optimized that can bind to PDDA and keep the AuNPs red in color. For this, different concentrations of T-Apt including 0.05, 0.1, 0.25, 0.5, 1, 2 and 3 µM , were added to a fix concentration of AuNPs(10 nM) in ultrapure water. After 20 min, 20 nM PDDA was added to the solution, and after incubation for 5 min uv-vis spectrum was recorded. As clear from the figure (Fig. 2S), 0.5 µM T-aptamer was successfully able to hybridize with PDDA and prevented the aggregation of AuNPs thereby maintaining the red color of the AuNPs. Optimization of reaction conditions causes set up a highly sensitive aptasensor for AMP.

3.2.2. Optimization of the reaction conditions for adsorbed polyA Apt colorimetric assay

polyA Apt concentration was optimized that can bind to PDDA and keep the AuNPs red in color. For this, different concentrations of polyA Apt including 0.05, 0.1, 0.5, 1, 1.5, 2 and 2.5 µM, were added to a fix concentration of AuNPs(10 nM) in ultrapure water. After 20 min, 20 nM PDDA was added to the solution, and after incubation for 5 min A680/520 was recorded. As clear from the figure (Fig. 3S), 1 µM polyA Apt was successfully able to hybridize with PDDA and prevented the aggregation of AuNPs thereby maintaining the red color of the AuNPs.

3.3. Colorimetric detection of ampicillin

AuNPs remain dispersed by electrosteric stabilization in aqueous solution. Generally, the positively charged material, such as cationic polymer and high concentration of sodium, can disturb the charge balance of the AuNPs and cause them aggregate. Herein, cationic polymer PDDA was employed to aggregate AuNPs. In this work, two types of ssDNA-functionalized AuNPs, T-Apt and polyA Apt, were synthesized through self-assembly. They are attached to AuNPs from 5′ terminal and 3′ terminal sequences, respectively. In the absence of AMP, the aptamer is hybridize to form a duplex with the cationic PDDA owing to the interaction of negatively charged phosphate backbone of aptamer with PDDA. Therefore, the aggregation of AuNPs is prevented , due to the lack of sufficient PDDA. However, upon the addition of AMP, the aptamer transformed to the secondary structure and forms a complex with AMP which makes the PDDA free and results in the aggregation of AuNPs. Accordingly, the significant change in the color of the AuNPs from red(λmax=520 nm) to blue(λmax=680 nm) is evident from naked eyes. The color of the solution is dependent on the concentration of PDDA which is directly linked to the concentration of AMP. Hence, the present two methodology including adsorbed T-Apt and polyA Apt on AuNPs can be employed for detecting of ampicillin colorimetrically.

After the careful optimization of all the reaction conditions for both methods, to evaluate the sensitivity of the present biosensor, employing UV–vis spectroscopy, TEM and visual detection. Different concentrations of AMP from 0-600 nM were added into the sensing solutions, and subsequently the UV–vis measurements were recorded. Fig. 2(adsorbed T-Apt method) and Fig. 3(adsorbed polyA Apt method) Clearly shows that upon increase in AMP concentration and , the characteristic peak of the AuNPs at 520 nm decreased while a new band emerged around 680 nm representing the aggregation of AuNPs. Accordingly, The color of the AuNPs dispersions went an obvious change from red(dispersed mode) to purple to blue(aggregated mode). In order to quantify AMP, ratio of the absorbance of aggregated peak to the characteristic AuNPs peak i.e. A680/520 was plotted and a calibration curve was obtained that fitted best to the concentration of AMP. In adsorbed T-Apt method, Fig. 2(b) shows that A680/A520 is proportional to the concentration of AMP with a regression coefficient R=0.991. Similarly, in adsorbed T-Apt method, Fig. 3(b) shows that A680/A520 is proportional to the concentration of AMP with a regression coefficient R=0.981. The detection limit of the aptasensor came out to be 0.1 nM and 0.49 nM in adsorbed T-Apt and adsorbed polyA Apt methods respectively, which was calculated using the formula 3α/slope [51, 52]. where α represents the standard deviation of the instrument and slope is obtained from the linear calibration plot. The proposed methods was linear in the concentration range of 1–600 nM and 1-400 nM in adsorbed T-Apt and adsorbed polyA Apt designs, respectively. Also based on visual detection, in adsorbed T-Apt and polyA Apt strategy, AMP in the concentration of 10 and 50 nM were detected by the naked eye, respectively. Compared with the reported methods for ampicillin detection, this proposed bioassay are relatively high compared to those of a previously reported optical and electrochemical biosensor, which was shown in Table 1.

The aggregation behavior of AuNPs was further verified by analyzing the samples through TEM and confirm the principle of this aptasensor. As shown in Fig. 4(a), the gold nanoparticles are well dispersed in their natural state and uniformly distributed and spherical in nature. The addition of an optimized concentrate of PDDA, causes electrostatic interactions between PDDA and AuNPs resulted in the aggregation of the particles thereby rendering the solution blue color [Fig. 4(b)]. However, in the presence of T-Apt and PolyA Apt, the PDDA formed a duplex structure with aptamer and therefore, the dispersity of the particles was maintained due to lack of sufficient PDDA and the solution remained red in color [Fig. 4(c), 4(d)]. Upon the addition of AMP to solution, due to high affinity of aptamer(T-Apt and PolyA) to AMP, PDDA again becomes free and lead to aggregation of AuNPs by electrostatic intraction into larger clusters. Gold nanoparticles capped whit citrate anion, maybe electrostatic attraction between cationic PDDA and citrat anion lead to come close of AuNPs and aggregated. As a result of this interaction, color of solution change to blue [Fig. 4(e), 4(f)].

3.4. Selectivity of the assay

The selectivity of the biosensor for AMP detection was also investigated. For this, A variety of competing non-target antibiotics, including amoxicillin, penicillin, benzylpenicillin and lincomycin were individually added to the sensing solutions, and the variations of absorbance values (ΔA) were calculated. Results [Fig. 5(a), (b)] and (Fig. 6(a), (b)] showed that changes in color from red to blue and variations of absorbance values (ΔA), was observable only for AMP. The biosensor works specificly only with ampicilin and gives a almost insignificant response to other antibiotics. Moreover, both designs assay that use of T-Apt(Fig 5) and polyA-Apt(Fig 6) in construction of aptasensor do not significantly differ in the specific recognized to ampicillin.

4. Conclusion

In summary, A colorimetric aptasensor for ampicillin detection has been successfully developed based on the aggregation of AuNPs controlled by the interactions among ampicillin, ampicillin aptamer and PDDA, with high selectivity. In this comparative study the role of two adsorbed Apt methods(T-Apt and PolyA Apt) on AuNPs surface on performance of colorimetric aptasensor to detect AMP were investigated. In adsorbed T-Apt ampicillin colorimetric assay detection of ampicillin was linear in the concentration range 1-600 nM with 0.1 nM as the limit of detection and in adsorbed polyA Apt ampicillin colorimetric assay was linear in the concentration range 1-400 nM with 0.49 nM as the limit of detection. Also based on visual detection, in adsorbed T-Apt and polyA Apt strategy, AMP in the concentration of 10 and 50 nM were detected by the naked eye, respectively. However in design and construction of many aptasensors thiolated modified aptamer were used, the results of our work clearly showed that compared whit thiolated modified aptamer, polyA unmodified aptamer can be used in the preparation of aptasensors with comparable sensivity and performance. Therefore, in construction of aptasensors, the polyA Apt can be used as a substitute for the T-Apt to measure a lot of analytes with satisfactory sensitivity.